Total RNA was prepared using Reliaprep RNA Miniprep kit (Promega). Ribosomal RNA was depleted using Illumina's Ribozero HMR kit, according to the manufacturer's instructions. Libraries were prepared using the KAPA Stranded mRNA-Seq Kit (Kapa Biosystems, Roche) on an Agilent Bravo liquid handling system and MJ thermocyclers. Libraries were sequenced on HiSeq 2500, single end 50 bp reads.
Ribozero hmr kit
Manufactured by Illumina
Sourced in United States
The Ribozero HMR Kit is a laboratory equipment product designed to enable the removal of ribosomal RNA (rRNA) from RNA samples. The core function of this kit is to facilitate the depletion of rRNA, which is a common step in RNA-sequencing workflows to enhance the detection and analysis of other RNA species of interest.
Automatically generated - may contain errors
Lab products found in correlation
Kapa stranded mrna seq kit, by Roche (1 mentions)
Reliaprep rna miniprep kit, by Promega (1 mentions)
Bcl2fastq conversion software v2, by Illumina (1 mentions)
Truseq stranded mrnaseq sample prep kit, by Illumina (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Hiseq platform, by Illumina (1 mentions)
Spriselect beads, by Beckman Coulter (1 mentions)
4 protocols using ribozero hmr kit
1
Total RNA Extraction and Sequencing
2
RNA-seq Analysis of Oocyte Fractions
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For RNA-seq analysis, each sample contained three biological replicates. Oocytes and eggs were fractionated as described the above. Before RNA extraction, a mixture of spike-in RNAs (egfp – 100 pg, mCherry – 10 pg, mRuby2 – 1 pg, firefly – 0.1 pg, renilla – 0.01 pg) were added into each 100 μL of the cytosolic, ER, and pellet fraction samples. Total RNA was extracted using TRIzol reagent according to the manufacturer’s instructions. Before library construction, ribosomal RNA was removed with the Ribozero HMR kit (Illumina), and the RNA libraries were prepared using the TruSeq Stranded mRNA-seq Sample Prep kit (Illumina). The libraries were quantitated by qPCR and sequenced on one lane for 101 cycles from each end of the fragments on a NovaSeq 6000 using a NovaSeq S1 reagent kit. FASTQ files were generated and demultiplexed with the bcl2fastq Conversion Software v2.20 (Illumina). Ribosomal RNA depletion, library construction, and generation of raw data were performed by the University of Illinois Roy J. Carver Biotechnology Center Sequencing Core Facility.
3
RNA Extraction from Tissue Samples
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Trizol (Invitrogen, USA) was used for extraction of total RNA from LA and control PWB according to manufacturer's instructions. The RNA concentration and purity were checked by OD A260/A280 (≥1.8) and A260/A230 (≥1.6), and the yield and quality were assessed using an Agilent2100 Bioanalyzer (Agilent Technologies, USA) and Ribo-Zero H/M/R Kit (Illumina, MRZH11124). The RNA integrity number (RIN) of extracted RNA was >7.0
4
Whole Transcriptome RNA-Seq Library Preparation
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High‐throughput whole transcriptome RNA‐sequencing (RNA‐seq) was performed by CD Genomics (Shirley, NY, USA). First, the total RNA was depleted of ribosomal RNA (rRNA) using the Ribo‐Zero HMR kit (Illumina, San Diego, CA, USA) according to the manufacturers instructions. The rRNA‐depleted RNA was then purified using 2x RNAClean XP beads (Beckman Coulter, Brea, CA, USA) followed by first and second‐strand synthesis using NEBNext, New England Biolabs reagents. The generated cDNA was purified with 1.8x SPRIselect Beads (Beckman Coulter). Endprep reaction was performed following the manufacturer's protocol for NEBNext Ultra II End Prep Reaction for the rest of the procedure. Adaptor ligation was then performed using NEBNext Ultra II Ligation protocols (New England Biolabs, Ipswich, MA, USA), and the ligated product was purified by SPRIselect Beads (Beckman Coulter) followed by elution in nuclease‐free water. PCR was carried out using NEBNext Ultra II Q5 Master Mix, and primers and the final library was subsequently purified with SPRIselect Beads. Libraries were sequenced using the Illumina HiSeq platform, generating 2 × 150 bp paired‐end reads and at least 50 million reads per library according to the manufacturer's instructions.
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